Journal: eLife

Article Title: Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62

doi: 10.7554/eLife.17290

Figure Lengend Snippet: ( A ) Scramble, MTOR and PIK3C3 sgRNAs were introduced into H4 Cas9 GFP-SQSTM1 cells by lentiviral infection and GFP fluorescence was analyzed after 7 days. Representative images show nuclei (blue) and GFP-SQSTM1 (green). Scale bar corresponds to 20 µm. ( B ) Pooled screening workflow of GFP-SQSTM1 assay. H4 Cas9 GFP-SQSTM1 cells are transduced with lentiviral libraries of sgRNAs or shRNAs and selected for stable integration. Fluorescence activated cell sorting (FACS) is used to isolate cell populations based on GFP upper quartile fluorescence (GFP high) or GFP lower quartile fluorescence (GFP low). A representative GFP FACS histogram is shown in . Abundance of sgRNA and shRNA sequences is quantified by deep sequencing of the corresponding barcodes in the genomic DNA of the isolated cell populations as well as unsorted cells. ( C ) Distribution of individual sgRNAs targeting MTOR, PIK3C3, PLK1 or SQSTM1 at day 7. GFP-SQSTM1 modulation was assessed as log2 fold ratio of each sgRNA sequence based on the abundance in the GFP high versus GFP low cell population. Anti-proliferative effects were assessed as log2 fold ratio of each sgRNA based on the abundance in unsorted cells versus the input library. ( D–F ) H4 Cas9 GFP-SQSTM1 cells were transduced with CRISPR or RNAi lentiviral libraries covering 2677 genes with an average of 20 sgRNA or shRNA reagents per gene. Both screens were run in duplicate and the mean is shown. Gene-centric visualization of ( D ) average log2 fold change (FC) or ( E ) redundant siRNA activity (RSA) scores in GFP high versus GFP low cells. Selected autophagy and MTOR pathway components are highlighted in red. ( F ) Log2 fold change (FC) distribution of all shRNAs and sgRNAs in GFP high versus GFP low cells. DOI: http://dx.doi.org/10.7554/eLife.17290.002

Article Snippet: For the focused libraries, custom sgRNA and shRNA libraries were constructed by Cellecta as previously described ( ).

Techniques: Infection, Fluorescence, Transduction, FACS, shRNA, Sequencing, Isolation, CRISPR, Activity Assay

Journal: STAR Protocols

Article Title: Scalable CRISPR-Cas9 chemical genetic screens in non-transformed human cells

doi: 10.1016/j.xpro.2022.101675

Figure Lengend Snippet: Next-generation sequencing data quality control (QC) plots Representative Illumina NextSeq 550 QC data. Three replicates of the DMSO vehicle control and Compound X screen from T0 and T3 are shown. (A) The number of reads per sample. Note that each sample has more than 3 million reads, which is the expected read counts for the 1000× coverage of the custom sgRNA library. (B) The mean base quality score for each position. The higher the score, the better the base call. Note that the sgRNA position is between 20-40 bp, which show very good quality in all samples. Green: very good quality (>Q28); yellow: reasonable quality (Q20-Q28); red: poor quality (

Article Snippet: Data for a bortezomib screen in hTERT RPE-1 cells using a custom Cellecta sgRNA library targeting 350 DNA damage response genes can be found here .

Techniques: Next-Generation Sequencing, Control

Journal: STAR Protocols

Article Title: Scalable CRISPR-Cas9 chemical genetic screens in non-transformed human cells

doi: 10.1016/j.xpro.2022.101675

Figure Lengend Snippet: PEI/DNA transfection mixture (for 3 × 15-cm plates, 10 million cells per plate)

Article Snippet: Data for a bortezomib screen in hTERT RPE-1 cells using a custom Cellecta sgRNA library targeting 350 DNA damage response genes can be found here .

Techniques: Transfection, Plasmid Preparation

Journal: STAR Protocols

Article Title: Scalable CRISPR-Cas9 chemical genetic screens in non-transformed human cells

doi: 10.1016/j.xpro.2022.101675

Figure Lengend Snippet:

Article Snippet: Data for a bortezomib screen in hTERT RPE-1 cells using a custom Cellecta sgRNA library targeting 350 DNA damage response genes can be found here .

Techniques: Western Blot, Recombinant, Modification, Lysis, Transfection, DNA Purification, Purification, Gel Extraction, Plasmid Preparation, Software, Picogreen Assay

Journal: STAR Protocols

Article Title: Chemical-genetic CRISPR-Cas9 screens in human cells using a pathway-specific library

doi: 10.1016/j.xpro.2021.100685

Figure Lengend Snippet: General workflow of a chemical-genetic CRISPR-Cas9 screen Can be adapted for use with another sgRNA library or cell type. Length of selection and treatment with compounds of interest will vary with the cell line, as will the number of days between time points during compound treatment. Sufficient sgRNA coverage must be maintained at each split and for each collection. “Pre-dox”, samples with integrated sgRNAs but before induction of Cas9. The “pre-dox” samples are essential for later data analysis. “Post-dox”, samples with integrated sgRNAs and after three days of Cas9 induction with doxycycline. Collecting the “post-dox” samples is optional.

Article Snippet: CRITICAL: When using a custom sgRNA library for the first time, you should also sequence the library itself using a unique Illumina indexing primer.

Techniques: CRISPR, Selection

Journal: STAR Protocols

Article Title: Chemical-genetic CRISPR-Cas9 screens in human cells using a pathway-specific library

doi: 10.1016/j.xpro.2021.100685

Figure Lengend Snippet: Example PCR products for sequencing library preparation 1.4% agarose gel of a sub-set of PCR products from replicates 1 and 2 treated with one of several compounds (A-M) for 8 days. UT, untreated. Pre, pre-dox. Post, post-dox. Each lane has 5 μL of a 300 μL reaction that began with 30 μg of genomic DNA, and each was amplified with a unique 5′ adaptor primer with unique index. “No template control” and “Negative control DNA” lanes each contain 10 μL of a 100 μL reaction. “Negative control DNA” reaction used 10 μg of genomic DNA from HAP1-Cas9 cells not infected with the sgRNA library.

Article Snippet: CRITICAL: When using a custom sgRNA library for the first time, you should also sequence the library itself using a unique Illumina indexing primer.

Techniques: Sequencing, Agarose Gel Electrophoresis, Amplification, Negative Control, Infection

Journal: STAR Protocols

Article Title: Chemical-genetic CRISPR-Cas9 screens in human cells using a pathway-specific library

doi: 10.1016/j.xpro.2021.100685

Figure Lengend Snippet:

Article Snippet: CRITICAL: When using a custom sgRNA library for the first time, you should also sequence the library itself using a unique Illumina indexing primer.

Techniques:

Journal: STAR Protocols

Article Title: Chemical-genetic CRISPR-Cas9 screens in human cells using a pathway-specific library

doi: 10.1016/j.xpro.2021.100685

Figure Lengend Snippet:

Article Snippet: CRITICAL: When using a custom sgRNA library for the first time, you should also sequence the library itself using a unique Illumina indexing primer.

Techniques:

Journal: STAR Protocols

Article Title: Chemical-genetic CRISPR-Cas9 screens in human cells using a pathway-specific library

doi: 10.1016/j.xpro.2021.100685

Figure Lengend Snippet:

Article Snippet: CRITICAL: When using a custom sgRNA library for the first time, you should also sequence the library itself using a unique Illumina indexing primer.

Techniques: Transfection

Journal: STAR Protocols

Article Title: Chemical-genetic CRISPR-Cas9 screens in human cells using a pathway-specific library

doi: 10.1016/j.xpro.2021.100685

Figure Lengend Snippet:

Article Snippet: CRITICAL: When using a custom sgRNA library for the first time, you should also sequence the library itself using a unique Illumina indexing primer.

Techniques: Recombinant, Subcloning, Plasmid Preparation, Purification, Transfection, Sequencing, Amplification, Software

Journal: STAR Protocols

Article Title: Chemical-genetic CRISPR-Cas9 screens in human cells using a pathway-specific library

doi: 10.1016/j.xpro.2021.100685

Figure Lengend Snippet:

Article Snippet: CRITICAL: When using a custom sgRNA library for the first time, you should also sequence the library itself using a unique Illumina indexing primer.

Techniques: Transfection